Preparation of cobalamins



April 1s, 1961 D. PERLMAN 2,980,591

PREPARATION OF COBALAMINS Filed Jan. 22, 1957 DAVID PERLMAN 3mm i such -as corn steep liquor .and grain mashesg"(3).-poly;v hydric alcoholsjsuch as glycerol; (4) fats, ,s\`1lch'asy 2,980,591 PREPARATION OF COBAIJAMINS Princeton, NJ., assgnor to Olin Mathieson Chemical Corporation, poration of Virginia Filed Jan. 22, 1957, Ser. No. 635,299

13 Claims. (Cl. 19E-65) This invention relates to an improved process for preparing physiologically active cobalamins:

Prior to this invention, cally active cobalamins could be prepared biosynthetically by culturingcertain microorganisms in a nutrient medium containing a precursor, the` nature of the precursor being determined by the particular cobalamin product desired. Thus, in the preparation of vitamin B12 [i6-dimethylbenzimidazole-cyano-cobalamin, employing the nomenclature of .Bernhauer et al., Angew. Chemie, 66, 776 (1954)] by the cells of -a microorganism such as Escherichia coli in a nutrient medium, the two precursors, 5,6-dimethylbenzimidazole and Fords factor B had to be added to the medium [Ford'et al., Biochemical Journal, 59, y86 (1955)].

This known process suiered the disadvantages of being relatively expensive due to the inherent inetliciency of the fermentation process, as a ylarge excess of precursors had to be utilized.

It is the object of this invention, therefore, to provide an improved process for the preparation of physiologically active cobalamins-which is more eicient andmore adaptable than those` priorly known.

These objects are achievedbythef'process of this-'inl'.

vention, which essentially comprises culturing a precursorrequiring vitamin B12-producing microorganism (particularly precursor-requiring vitaminY B12-producing microorganisms of the genus Propionbacterium) in a-cobaltcontaining nutrient medium substantially free of precursor, separating the cells fromithe medium, treating, the separated cells with a precursor and recovering the' resulting physiologically active cobalamin.

Among the microroganisms which may be employed in the practice of this invention are those known to prod'uce vitamin B12 in the presence of a 5,'-6-dimethylbenzimidazole-containing nutrient medium but which fail to elaborate it was known that physiologinited States Patent O and tripalmitin; and fatty acids having more than 14 carbon atoms, such as stearic acid, palmitic acid,

oleic acid, linoleic acid and myristic acid.

' fnitrategor itrmayl beprovided in the form of organicallyvbound cobalt, such as acobalt-containing yeast, preferably'one containing a relatively high proportion of cobalt (i.e., about 100 or more parts per million). The nutrient media may, of course, contain any of the j additional components usually found in such solutions (except, of course, a precursor yfor the cobalamin desired); among theseaddit'ional components are antifoam j agents (e.g.,'lard oil, octadecanol, etcL), metallic cations,

such as potassium, calcium, magnesium and iron (which may be present in the crude materials used in the nutrient v medium), andv phosphates (which may be added as in'- .30 tions, a source of oxygen or air should also be present.

organic phosphate).

The fermentation process may be carried out `at tem- If the This aeration can be accomplished by bubblingv air (or oxygen) through the medium during the fermentation or v by agitating the medium, thereby exposing a large sur- "face thereofto the atmosphere. 2If thev microorganim -is anaerobic inl nature, of course, this aeration step must then be omitted.

After a suflicient incubation time (about one to ten 1 days), the medium is separated into its liquid and solid :f componentsVas by-iiltrationzory centrifugation, and the cells are suspended in water (preferably distilled water).

This suspension can then, if desired, be again separated into its solidand liquid portions and the solid component resuspended ,in water. The pH of the iinal suspension usually is slightly acidic (i.e., pH less than seven). If it is not, Ian acid such asdililte hydrochloric acid is added f justed suspensionyis one in the` range' of vabout 5fto the vitaminv Whenlthe nutrient medium is substantiallyV free from the precursori-Examples of Vsuch microorganisms include variousspecies of Propionbacterium, -as exemplifiedby Propionibacterum ambinosum, Proponbacterumpentosaceum, Proponibacterium zeae, Proponz'bacterz'um zhoenz'i,` Proponibacterium Yrubrum and Propz'onz'bacterum peterssoni.

ble starches; (2) substances containing carbohydrates,

lard oil, soybean oil,"h'nseed oil," cottonseedi oil',"peanut 011,

Vcoconut oil, corn oil, Icastor oil, sesame'oil, palm oil, -v mutton tallowfsperm Ao i1,.-.,olive oil, tristearin, triolein;

vabout, 7.4' To the suspensionis added at least the stoichiometrically required amount ofprecursor -to yield the del sired cobalamin, and the reaction is allowed to proceed (preferably, expedited by agitating the suspension) until optimum cobalamin formation has occurred, .The snspensionY isethen -heated"(to release'th-icohalamin from the cells), filtered or centrifuged, and the cobalamin recovered from the liquid portion. As an alternative procedure, .the cells after separation from the medium canV i be lysed as by treatment with asolvent such as acetone or n-propanol before the precursor is added. d v .The natureof.the;precursori employed `depends on the physiologically active cobalaminvdesired. Thus, if vitat min ,B12 is desired, 5,-dimethylbenzimidazole or another vitamin B12 `precursorpsuchas 2,3-dimethyl-5,6-diaminobenzene,2,3dinitrorS,-dimcthylbenzene or 2,3-dimethylt 4amino'5nitrobenzene, may be used; If an funnatural vitaminY B12 which possessesvitamin B12-like activity is 2 desired; aprecuisr-"ffor' thpart'iculai" cobalamin Acan be j .,use'd, .Thus, kas summarized in the following table, kthe Y nature ofthe resulting cobalamin will depend on the'pref Precursor Product (Y-eobalamlns) Name 2-Methyl-4-methyl-thioquinazo1ine 2-Mehy1-4-methy1-thloquinazoline-cobalamm.

Phenmi n n n N 2 Nitr0phenazine 2Nitrophenazinecobalamin N0:

2-Aminophenazine 2-Aminophenazine-cobalamin NH,

. l i N 1Amlino2hydroxyphenazine 1-Amino-Z-hydroxyphenzine-cobalamin il OH 1-.A.ce1:amido-3-methoxyphenazinef 1Acetamido-3methoxyphenazine-cobal- Z-Hydroxyphenazine Y2-12isfd1'oxyphenazin-cobalamin @70H n l. Y N t NR A C C-l-CHn' 5Methylbenzimidazole 5-Methylbenzimidazole-cobalamln C (ll3 C13H N \CH Quinoxalinm, A Quinoxalne-cobzalarrlin'. ..v s A In the drawing X representsvan anion,"for examlole, 'a hydroxy radical or theanion of an acid (preferably a ,pharmacologically acceptable acid). Examples ofY suitable 'anions are the anions lof mineral acids-(e'.g.,1clxloY ride, bromide,l sulfate, nitriie andwnitraffe), cyanide,f cyanate; etc. If no utilizable aniongisr present 'eiiher'in 'the fermentation'rnedium or"added with the prec'rsor in the'second step?. a hydroxy-cobalaminis initially formed (XIOH). lf, however,'thernedium containsaisonrce;

.of'aniorn such as cyanide, VLherrespctiye anionic-.cobala-f'" v min fs recovered as -the product (e.g'., X='CN);= Furtlermore, if a par'ticlar salt is desired, the hydroxy-cobala- .-min can be converted to thesaltvby tr'eatmntrwitvlian acidi' Thu'sv, a hydroiry-cobalamin; upon 'treatnfleritf with hydrochloijicfacidl., yieldsY tihe clilorideV (X= Cl) vors-With `V hydrogen cyanide :.(or potassium Vcyanide in an acidic v medium) yieldsithe cyanideKXzCNy i ,AlthoughfI-do not Wish. to be limited to any ktheoretical considerations, vit would appear'that theriniti'a-l fermentation step result-s in the formation `of a small yamount of a V.physiologically inactive adenine-'cobalamin` of the cobalamin molecule on the addition of the de sired precursor in the presence of the enzymatic vsystem of the washed cells of the microorganism. Thus, it would 'd' appear that .adenine-hydroxo-cobalamin is formed in the first step of Example A1. Upon addition of 5,'6-dimethylbenzimidzole to the aqueous suspension of the washed cells of Propionibrzcterum arabinosm, 5,-6-dimethylbenzimidazole-hydroxo-cobalarnin is formed. This can be converted to vitamin B12 by addition of potassium cyanide in the acidic medium.V It would further appear that the adenine-cobalamin is `introduced into the aqueous suspension bound to the cells of the microorganism and is subsequently released therefrom either by treatment of the suspension with an acid or because ofthe inherent acidity of the suspension.

To show the homogeneity and `activity of the cyanocobalamins formed in the examples of this invention, the following tests were conducted. For these tests the cyanocobalamin was dissolved Yin water at a concentration of about 100 micrograms of cyanocobalamin per ml. of water:

The solution of the cyanocobalamin is dried on a lter paper strip of Whatman 3 MM paper in parallel .with samples of 5,6-dimethylbenzimidazole-cyanocobalamin, adenine-cyanocobalamin, Z-methyl-adenine-cyanocobalamin and Fords factor B [Ford et al., Biochem. Jour. 59, 86 (1955 The sheet is placed in an ionophoresis apparatus [similar to that described by I-loldsworth in Nature, 171, 148 (1953)], `and the paper is impregnated with a solution of 0.5 N acetic acid containing 0.02% KCN (w./v.). A potential of about 280 volts is applied for about 17 hours. dried. When dry (and free from odor of acetic acid), it is applied forr v15 `minutes to the surface of an agar plate seeded with a suspension lof a vitamin B12-requiring strain of Escherichia coli (ATCC 1.1105). [The agar medium contains (grams/liter); sucrose, 20 g.; citric acid, 1.2 g.; (NHQQHPOQ :4 g.; KCl, 0.08

' 6H20, `lwil'lclg' i1-120,

0.036 gli The sheet is removed and plated on seeded agar plates as in Test I. growth are determined. v

` )b Same as Test Illa with 1.0 ml. of glacial acetic acid substituted for the yammonium hydroxide.

Zones 'of TEST IV An aliquot is assayed for the presenceof substances stimulatingy the growth of Lactobacillus `leichrmznn (ATCC 7830) using as standard 5,6-dimethylbenzimidaZole-cyanocobalamin and the method of the U.S. Pharmacopoeia (15th edition). A value is determined.

TEST V Exam pl e 1 VITAMIN Bm Anmedium containing 20 grams autolyzed yeast, 30 grams glucose, 25 milligrams Co(iNO3)2-6H2O and 1 liter of tap water is prepared, and SOO-ml. aliquots are placed in 1-liter Erlenmeyer .flasks The flasks are plugged vwith non-absorbent cotton and autoclaved at 121 C. for 3() minutes. Approximately 10 grams of powdered calcium carbonate (previously sterilized by heating for at 'least-3 Ahours inV a', oven maintained at 150) are added 0.04 g.; agar, 15 g.; triphenyl `tetrazolium chloride, .1 g.] Y After 18 hours incubation aat 37 C;, the agar plate is TEST 'Il An aliquot of the solution is applied to a spot about 3 inches from the end ofva strip of Wha-tman No. 1

filter paper parallel to spots of known cobalamins. The

chromatogram is developed bythe descending .method using a solvent mixture containing: 77 ml. of seo-butanol, 23 ml. of water, 0.25 ml. of `KCN solution (5 gms./ 100 ml.) and 100 mg. of KCIO.; for 24 hours (at 25 C.). i

The strip is dried and applied to the seeded agar plate as in Test 1..'After incubation, the Zones of growth, representing the, presence of vitamins of the IB15 group (measured with reference Vto the movement of 5,6-di

methylbenZimidazole-cyanocobalamin), aredeterrnined.V

TEST 11i) (a) A11 aliquot ofthe solution is applied to a yspot hof about 0.9 mg. pen'liter is obtained. Another aliquot.y approximately 10.3111. inrvolur'nefis shaken with 10 m1, of,avmixturesofffphenol and benzene (70 parts l88% about 3 inches from the end of-a strip of Whatman No.VV

4 filter paper parallel to spots of known cobalamins. The chromatogram isj developed by the descending` meth, od using a'solvent'mixture containing: seo-butanol, 1,00

` ml.; water, 50ml.; KCN `[5% solution (w./v.)], 0.25

ml.;fand VNHiQI-IV,(concentrated), 1.0 ml` After 17 l' vhm'rsf development (at 35 C.),"the strips are dried and vpH- 6.2. midazole is added yto one ofthe series of flasks, and thevv Pirenei-30, pats benzne).

to each flask.` When the liquid has cooled to 30, the asks are inoculated with 10 ml. of 2-day-old culture of Proponibacterum arabz'nasum ATCC 4965 (American Type CultureCollection, Washington, D.C.) grown on this medium. The flasks are thenrplaced on a reciprocating shaker (1201-inch cycles per minute), located ina constant temperatureroorn maintained at 30. After 4S hours to 74 hours incubation, the cells, debris and calcium carbonate in the ask are :collected by centrifueation.v The collected solids are resuspended ina volume of dis- Y tilled water equal to the original, shaken on a reciprocating shaker (2'80'1-inchcyclesper minute) lfor 5 minutes and recentrifuged. The washed solids are resuspended in dis- "lled water (equal in volume to half the original volume), and aliquots are distributed into flasks (20 ml. per 125- ml. Erlenmeyer flasks or square-base 6-oz. bottle is'a convenient volume). The pl-l of the suspension is about Approximately 2,00ug.` of,5,6dimethylbenzi flasks are placed on a reciprocating shaker (120 1inch cycles per minute), located in a room maintained at 30. After 20 hours agitation, the pH of the suspension rises to about 6.7. The contents ofthe flasksV are heated Vat to 90'. C. for" 20 minutes"`(in a'boiling water bath), andthe suspension is centrifuged. Approximately 0.5 m1. of an aqueous solution of KCN (5 g./ 100 ml.) is added to20 ml. of the supernatant liquid. An aliquot of this,v supernatant liquid (hereinafter referred to as the supernatantliquid) is analyzedV for the presence of sub- Y stances stimulating the growthof Lactoilmcillus lechmannii (ATCC 7830), using as standard 5,6;dimethylbenzimidazole-cyanocohalamin and .the methodiin ther-UfS. Pharmacopoeia (fifteenth edition) (Test IV). A value The mixture. is centrifuged,

and .the upper'layer (ca.V 8 Vroll) is Vtransferred 'to Ya 'test .tube.' YAn equal volumeof Yn-butanol is' addedvto'l this y phenol-benzene extractpand the solution is shaken brieiiy.l

Five-ml. of Wterlvis lthen added, the mixture shaken on ',a reciprocating Shaker .for 10l minutes vand.then-,c en` ftr'ifu'getl.l The 'bottom aqueous layer-(hereinafter called the aqueous concentrate) removed and analyzed by the following tests:

TEST I In this test the major cobalamins present in the aqueous concentrate has a mobility equal to that of 5,6-dimethylbenzimidazole-cyanocobalamin. There is also some adenine-cyanocobalamin present. A sample of the aqueous concentrate from a cell-suspension which had not been supplemented with the 5,6-dimethylbenzirnidazole shows only the presence of adenine-cyanocobalamin.

TEST II In this test the major cobalamin present in the aqueous concentrate has a mobility equal to that of 5,6-dimethylbenzimidazole-cyanocobalamin. There is also a cobalamin present which has a mobility about 0.35 that of the 5,6 dimethylbenzimidazole cyanocobalamin vor equal to that of adenine-cyanocobalamin. The aqueous concentrate from the unsupplemented cell-suspension shows only the presence of adenine-cyanocobalamin.

TEST IIIa In this test the major cobalamin present in the aqueous concentrate has a mobility equal to that of 5,6-dimethylbenzimidazole-cyanocobalamin. There is also a cobalamin present with a mobility about 0.6 that of the 5,6-dimethylbenzimidazole-cyanocobalamin. A sample of the aqueous concentrate from the unsupplemented .cell-suspension shows only the presence of adenine-cyanocobalamin;

TEST IIIb Porter [Brit I. Nutrition, 7, 326 (1953,)1 With'5,6di"

methylbenzimidazole-cyanocobalamin as standard. A value of about0.59 mg. per liter is obtained. When an V10 aqueous concentrate is found to contain 5,6-dimethylbenzmidazole-cyanocobalamin and a small amount of adenine-cyanocobalamin, as shown by the results of Tests I to V, described in Example 1.

The same results are obtained when 2,3-dimethyl-5,6 diamino benzene is substituted for the 5,6-dimethylbenzimidazole. Incubation of the cell-suspension under anaerobic conditions, e.g., under an atmosphere of nitrogen, did not aiect the biosynthesis of cobalamins, as determined by the bioassays and the response to Tests I .to V.

Example 3 BENZIMIDAZOLE-CY AN 0 COBALAMIN The procedure described in Example 1 is used with the replacement of the 5,6-dimethylbenzimidazole with benzimidazole. The bioassay of the supernatantliquid shows about 0.9 mg. per liter of activity by the L, leichmannii assay (Test IV) and 0.31 mg. per liter by the 0. malhamenss bioassay (Test V); in both assays, 5,6-dimethylbenzimidazole-cyanocobalamin is used as standard.- Analysis of the aqueous concentrate prepared as in Example 1 shows the presence of an ionophoretically neutral cobalamin (by Test I). The major cobalamin in the aqueous concentrate has a mobility about 0.9 that of 5,6-dimethylbenzimidazole-cyanocobalamin in Testv II, 0.95 in 0Test IIIa and 0.95 in Test IIIb..` These are approximately the values obtainedwith benzimidazole-cyanocobalamin in these tests. f Y

' The same results are obtained .when o-phenylene diamine orV O-dinitrobenzene is substituted fork the benzimidazole. l Y

' l yExample 4 Y BENZIMIDAZOIiE-CYANOCOBALAMI The procedure described in Example 2 is'used with the replacement of the 5,6-dimethylbenzimidazole withbenziinidazole. The bioassay of the supernatant liquid shows aliquot of the supernatantliquid lfrom an unsupplemented A,

f, :supernatant liquid'shows about 0.8 mg. per lterofactivity cell-suspension is assayed, a value of 0.01 mg. per liter,

is obtained.

Other substances which may be used instead of thej5,6 -dimethylbenzimidazole in the procedure of Example l include: 2,3dimethyl-5,6diaminobenzene; 2,3-dinitro5,6 Vdirnethylbenzene; or 2,3dimethyl-4-amino-S-nitrobenzene. The cell-Suspension may be shaken in an atmosphere of nitrogen instead of air without affecting the biosynthesis yof the cobalamn. Y l

Example, 2` VITAMIN B12v 'The same procedure as used inExamplevl is used 'with a culture of Propionbacterium pentosaceumvATCC4875 measured by the L. lechmnnii zole-cyanocobalamin as I 0.38 mg. per liter as determined bioassay (Test IV) and about 0.8 mg. per liter of activity by the L. lechmanmi assay and 0.27 mg; per liter by the O. mqlhamens'is bioassay (in bothfassays 5,6-dimethy1benzimidazole-cyanocobalamin is used as a standard). Analysis of the aqueous concentrate shows the presence of benzimidazole-cyanocobalamin, as ldescribed Example 3. n

Example 5 e-frarmuoRoMnTHYLBnNznurDAzoLELoraNoco BALAMIN v procedurevfused in Example 1 is used thereplacement of the 5,6.-dm`ethylbenzimidazole lwithva,a,avtriiuorornethyl-Z-nitro-p-toluidine The bioassay f ofvjthe by the L. leichma'nni assay and 0.19 mg. per-liter by the O. mulhamenss bioassay (in both assays 5 ,6-dimethy1benzimidazole-cyanocob'alaminy is used-as astandard); Analshows a cobalamin with a mobility of about 0.95that of .same result is obtained when analysis is made of the aque the 5g6-dimethylbenzimidazole-cyanocobalamin,fand the '..ousfconcentrat'ebythe procedures of Tests AIIIay and IlIb instead of the P. arabnosum. The supernatant liquid conf tains about 0.87 mg. per literV of 5',6-dimethylbenzirmda-I'l When 3,Lt-diamino-fx,,a-triuoromethyltoluene -issubstitilted for the lt1iflu0r0-2-nitro-p-toluidine, Lthe same results are'obtained; 'f i Theprocedurel described in :'Example vis used Vwith y `the replacement of the. 5,6-dimethylbenzimidazole with -baai,a-triiluoro-Z-nitro-p-toluidine.' v.supernatant liquid is 4about 0.7- mgfiper literas 1lreasured by thelflechmanni bioassay"(using 5,6-dim`ethylben- '-z'imida,zolecyanocobalamin as'v standard The bioassay of -'the Analysis-pf g same as before centrifugation.

. il Y theaqueous'` concentrate by the procedures of Tests rI to IIIb givesthe'same results described in Example 5.

Example 7 -METHYLBENZIMUDAZOLE-CYANO COBALAMIN The procedure of Examplel is used with the replacement of the 5,6-dimethylbenzimidazole with S-methylbenzimidazole. VThe bioassay of the supernatant liquid shows about 0.9 mg. `per liter 'as measured by the L. leichmanmi 'assay 'and 0.23.v mg.V per liter as measured by the O. malhamenss bioassay (in both assays 5,6-dimethylbenzimidazole-cyanocobalamin is used as a standard). Analysis of the aqueous concentrate by the procedure of Test I shows the presence of an ionophoretically neutral co- 'balamiIL Analysis of the aqueous concentrate bythe procedure of Test II shows the presence of a cobalamin with a mobility of about :88 that of 5,6-dimethylbenzi- 'midazole-cyanocobalamin, `while when the aqueous concentrate is Ianalyzed by the procedures of Tests IIIa and -IIIb,` the cobalamin has a mobility equal to that of the `5,6-dimetl1ylbenzimidazole-cyanocobalamin.

Example 8 QUINOXALINE-CYANOCOBALAMIN The4 ,procedureused in Example 1 is used-with the replacementfof 5,6-dirnethylbenzimidazole with quinox- Valine hydrochloride. The bioassay of the supernatant liquid is approximately 0.8 mg. per lliter when-measured vbythe growth response of L. leichmfmnz' (with 5,6-dimethylbenzimidazole-cyanocobalamin as standard). Examination of the aqueous concentrate by the procedure of Test I shows an ionophoretically neutral cobalamin. When the aqueous concentrate is analyzed by the procedure of .'1`e's-tILa cobalarnin 'with :a mobility of 0.86 that kof 5,6-dimethylbenzimidazole-cyanocobalamin is found. VWhen the aqueous concentrate is analyzed by the procedure of Test IIIb, a new cobailamin witha mobility -of 0.9 thaty of the..5,-dimethylbenzimidazole-cyanokcobalamin Vis found, while when theprocedure of Test IIIa Vis yused, the `new cobalarnin has `a mobility of 0.7 l.that of the 5', 6.dimethylbenzimidazole-cyanocobalamin.

vEixample 9v Y 2-HYDROXYPHENAZINE-CYANOCOBALAMIN A .culture ofV Propoz'bacterum ambnosumi (obtained asin Example Vl) is "grown on Athe iautolyzed yeast-glucose- Co(NO3)2-6H2O- CaCO3 medium of Example l fo-r two daysI (Without the inclusion Vin the medium of'a phenazine precursor).V At the end ofv this period, the solids (cells, debris Vand CaCO3) are separated from ,the liquid by centrifugation 4and resuspended in -a volume of distilled r -vwate'r so that the cell concentration is approximately the The suspension is well agitatedand the solids collected again by centrifugation. These solids are then resuspendedin distilled water equiv- V`alenttohalf the volume before centrifugation. Aliquots of this suspension are distributed into flasks (20 per 12S-ml.- Erlenmeyer flask .is a convenient'volume), vand 1 l ml. of an aqueous solution of `2phenazinol is added to standard).

Example 10 BENZOTRIAZOLE-CYANO COBALAMIN The procedure of Example 1 is used with the replacement of l5,6-dimethylbenzimi'dazole with benzotriazole. The bioassay of the supernatant liquid is 0.9 mg. per liter as measured by the L. leichmannii method and 0.3 mg. perliter as measured by the O. malltamenss bioassay (using 5,-dimethylbenzimidazole-cyanocobalamin as a Analysis of the aqueous concentrate by the procedure of Test I shows the presence of an ionophoretically neutral cobalamin. Analysis of the aqueous concentrate by the procedure of Test Il shows the presence of a cobalamin with a mobility of 1.05 that of 5,6-dimethylbenzimidazole-cyanocobalamin, while when the procedure of Test IIIb is used, the new cobalamin has a -mobility equal r tov `that of 5,6-dimethylbenzimidazolecyanocobalarnin used,- the new cobalamin 'has a mobility of about 0.3 thatr When the procedure of Test IIIa is of 5,-dimethylbenzimidazole-cyanocobalamin.

\mins formed in each of the examples can bev used in lieu of otherwise-produced vitamin B12 in promoting growth The cyanocobalamins formed in each of the examples can be converte-d to the corresponding hydroxocobalamin derivatives by treatment of the former with hydrogen in the presence Vof platinum oxide in an aqueous medium. The hydroxocobalamins, thus formed, can then be converted to any desired salt by treatment with the appropriate acid in an aqueous medium.

The vitamin B12 and other biologically active cobalaof chicks. For this purpose, the cobalamin-eontaining supernate maybe merely dried, to provide a cobalamin concentrate;A or the cobalamin may be recovered from Vlthe'supernate ordn'ed concentrate by use of conventional vitamin B12 purioation expedients.- The -dosage erniployed (e.g., when added as a supplement-to chick feeds) would depend on the Apotency of the concentrate, or

one ofthe groupsv so that the concentrationzof phenazinofl` is approximately 10mg. per liter. These asksare placed on areciprocatin'g shaker (120 l-inch strokes per minute), located in a room maintained at 30. After 18 hours shaking, 0.5 ml. of 5%V KCN is added, and the contents of the asks areY heated at 85 yto 90 for 30 minutes ina boiling water bath; VThe solids are collected by centrifugation `and Vthe supernatant liquid extracted with" arphenol-benzene solution as inExample 1. An aliquot `ofthe'supernatant solution,l when assayed by the L. leichmanm' assay(see Example 1),.gives avalue of 1.03 'y/ml.; and A.whenftheOchromorms malhamenss assay is .used,fthe'value is V0.52 fy/nl.. The benzene extracts are'l *pooledV-an equal volume ctn-butanol `is added and the :mixture-extracted 174m its )volume .of water. 1: The

l is Propon'ibacterum' arabinosum. Y

. i `5. The process of claim 1, wherein the microorganismV potency of the isolated non-B12 cobalamin, relative to pure vitamin B12. The invention may be otherwise variously vembodied within the scopeof the appended claims.

I claim:

V1. A process for preparing a physiologically active cobalamin, which consists of culturing a precursor-requiring vitamin B12-producing microorganism in a cobaltcontaining nutrient medium substantially free .of precursor, separating the cells from the medium. to obtain resting cells, treating said separated resting cells with a precursor and recovering the resulting physiologically active oobalamin.

2. The process of claim l,I wherein the treatment Awith precursor is .conducted under acidic conditions.

3. The process of. claim 1, wherein the niicroorganism lisof..the` genus Propionibacterium. "4. The processjof claim 1, wherein the microorganism is Popz'onbacterium pentosacezfm.,

6.The process of claim` 1, wherein the precursor is .5,6k-dimethylbenzimidazole and a 5,6-dimethyl'benzimidazole-cobalaminis recovered.

7. 'The process Iofjclaim 1,whereinthe" precursor is v benzimidazole, and a rbenzimidazole-cobalamin is re-:

covered.

8. The process of claim 1, wherein the precursor is ,,a-triuoromethyl-2-nitro-p-toluidine, and a S-trifluoromethylbenzmidazole-cobalan'n is recovered.

9. The process of claim 1, wherein the precursor is 5-methy1benzin1idazo1e, and a 5-methylbenzimidazo1ecobalamin is recovered.

10. The process of claim 1, wherein the precursor is quinoxalne, and a quinoxaline-cobalamin is recovered.

11. A process for preparing a 5,6-dimethyl-benzimidazole-cobalamin, which consists of culturing a precursorrequiring vitamin B12-producing microorgansm of the genus Propionobacterium in a cobalt-containing` nutrient medium substantially free of precursor, separating the cells from the medium to obtain resting cells, treating said separated resting cells with 5,6-dimethylbenzimida zole under acidic conditions and recovering the resulting 5,6-dimethy1benzirnidazole-cobalamin.

14 12. The process of claim 11 wherein the microorganism is Propionibacterium arabiriosum.

13. The process of claim 11 wherein the microorganism is Propionbacterium pentosaceum.

References Cited in the file of this patent UNITED STATES PATENTS 2,715,602 Hargrove et al. Aug. 16; 1955 2,764,521 Leviton Sept. 25, 1956 2,842,540 Perlman July 8, 1958 2,893,988 Bernhauer et ral. July 7, 1959 OTHER REFERENCES Germany, A19703 Iva/30h, March 8, 1956. 

1. A PROCESS FOR PREPARING A PHYSIOLOGICALLY ACTIVE COBALAMIN, WHICH CONSISTS OF CULTURIING A PRECURSOR-REQUIRING VITAMIN B12-PRODUCING MICROORGANISM IN A COBALTCONTAINING NUTRIENT MEDIUM SUBSTANTIALLY FREE OF PRECURSOR, SEPARATING THE CELLS FROM THE MEDIUM TO OBTAIN RESTING CELLS, TREATING SAID SEPARATED RESTING CELLS WITH A PRECURSOR AND RECOVERING THE RESULTING PHYSIOLOGICALLY ACTIVE COBALAMIN. 